Aromatic hydrocarbons are used in many aspects of present day technology. They form the basic constituents of fuels and are used extensively for the synthesis of drugs, herbicides, pesticides and many other commercial chemicals. They are also pollutants of urban environments. This proposal is directed towards elucidating the mechanisms utilized by bacteria to initiate the biodegradation of aromatic hydrocarbons. Pseudomonas putida oxidizes toluene to (plus) minus 1 (S), 2(R)-dihydroxy-1.2-dihydrotoluene. Both oxygen atoms in the hydroxylated product are derived from a single molecule of molecular oxygen. The enzyme system (toluene dioxygenase) will be purified. The properties of each individual protein components will be determined. A reconstituted system prepared from homogeneous components of the toluene dioxygenase complex will be used to investigate the mechanisms of substrate-oxygen-enzyme interactions. Techniques to be utilized include salt and solvent fractionation, ion exchange chromatography, preparative electrophoresis, affinity chromatography, stop-flow spectrophotometry, electron spin resonance spectrometry, optical rotary dispersion and circular dichroism spectrophotometry. Similar investigations will be performed with naphthalene dioxygenase, an enzyme in Pseudomonas sp. NCIB 9816. This multienzyme system oxidizes naphthalene to (plus) -1(R),2(S)-dihydroxy-1,2-dihydronaphthalene. The reactions utilized by bacteria to oxidize aromatic hydrocarbons are different to those utilized by mammals. Enrichment culture techniques will be utilized to isolate eucaryotic microorganisms that degrade toluene and naphthalene via an initial monooxygenase enzyme system.